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EXO1 Plays a Carcinogenic Role in Hepatocellular Carcinoma and is Related
to the Regulation of FOXP3
ABSTRACT
Exonuclease 1 (EXO1), a member of the RAD2 nuclease family, was first
described as possessing 5′ to 3′ nuclease activity and 5′ structure-specific
endonuclease activity. Here, we show that EXO1 is significantly upregulated in HCC
tumor tissues and that high EXO1 expression is significantly correlated with liver
cirrhosis. We further demonstrate that EXO1 knockdown decreases proliferation and
colony forming abilities of HCC cells in vitro and tumorigenicity in vivo, as well as
decreases migration and invasive capabilities of HCC cells. Alternatively, EXO1
overexpression significantly increases the proliferation, colony forming ability, and
migration and invasive capabilities of HCC cells in vitro. Additionally, we truncated a
region upstream of the transcription start site (TSS) of EXO1 and used the region with
the strongest transcriptional activity to predict that the transcription factor FOXP3 can
bind to the EXO1 promoter. Bioinformatics analysis found that FOXP3 was positively
correlated with EXO1 and luciferase reporter assays and RT-PCR confirmed that
FOXP3 could enhance the transcriptional activity of EXO1. CCK-8 assays showed
that depletion of FOXP3 further reduces cell proliferation ability after knocking down
of EXO1 in vitro. Taken together, our findings indicate that EXO1 acts as an oncogene
in HCC and its expression level is related to FOXP3 activity.
Key words:
EXO1, FOXP3, Hepatocellular carcinoma, Metastasis, Transcriptional regulation
Introduction
Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third
leading cause of cancer-related deaths worldwide [1]. Most patients are initially
diagnosed at an advanced stage in the disease and thus, the curative effect of
hepatectomy is often unsatisfactory. The 5-year recurrence rate of HCC after
hepatectomy exceeds 80%, and the 5-year survival rate is only 30%-70% [2, 3].
Therefore, in-depth study of the molecular mechanisms involved in the development
of HCC is of great significance to develop effective therapeutic drugs and prevent the
recurrence of HCC after hepatectomy.
Exonuclease 1 (EXO1) is a member of the Rad2/XPG family of nucleases and was
originally identified in Schizosaccharomyces pombe [4]. EXO1 possess 5' to 3'
exonuclease activity and structure-specific endonuclease activity and plays an
extremely important role in biological processes such as DNA replication, DNA
mismatch repair (MMR), DNA double-strand break repair (DSB) and telomere
maintenance [5-9]. Deletion of the EXO1 gene causes genomic instability and leads to
impaired DNA damage repair and meiosis defects [7, 10-13]. However, studies have
shown that abnormally high expression of EXO1 is related to the occurrence,
development and prognosis of various malignant tumors. Kretschmer et al. reported
for the first time that EXO1 is significantly elevated in breast ductal carcinoma and
invasive ductal carcinoma [14]. A recent study by de Sousa et al. has shown that
EXO1 expression is significantly elevated in glioma, and high EXO1 expression is an
independent risk factor for poor prognosis of patients [15]. Dai et al. reported that
EXO1 is upregulated in HCC specimens and EXO1 overexpression may also be
associated with poor prognosis in HCC patients [16]. In addition, several single
nucleotide polymorphisms (SNPs) in the EXO1 gene are associated with tumor
susceptibility in a variety of tumors including liver, gastric, ovarian, cervical, breast,
and colorectal cancers as well as head and neck squamous cell carcinoma [17-25].
Among them, rs1047840 (K589E), a common SNP genotype, increases the risk for
developing non-viral liver cancer, colorectal cancer and lung cancer [17, 26, 27].
However, the role of EXO1 in the invasion and metastasis of HCC and the upstream
transcriptional regulation of EXO1 is still poorly understood.
Forkhead box P3 (FOXP3) is a member of the forkhead/winged-helix family of
transcription factors, which was first identified in T-regulatory (Treg) cells as an
essential protein for regulating immune system development and function [28].
FOXP3 is primarily known for its function in regulating the CD4+CD25+ regulatory
cells and for defining their immunoregulatory phenotype [29]. Like other transcription
factors, FOXP3 can bind to numerous enzymes and microRNAs to up- or
downregulate a large number of genes [30]. Dysregulation of FOXP3 has been
reported in the context of various tumors types indicating that it could be a poor
prognostic factor in colorectal cancer and bladder cancer [31, 32], but a potential
tumor suppressor gene in breast cancer [33, 34]. Nevertheless, whether FOXP3 can
regulate the transcription of EXO1 is poorly understood.
In this study, we discovered that EXO1 expression is significantly higher in HCC
tumor tissues. It was also found that EXO1 could promote the proliferation of HCC
cells in vitro and in vivo, as well as promote the migration and invasion of HCC cells
in vitro. FOXP3 could lead to the upregulation of EXO1 at the transcriptional level,
where it could act as an oncogene in HCC as well. Our research revealed the
relationship between the abnormal expression of EXO1 and malignant biological
characteristics of HCC and might lead to the development of novel anti-cancer
therapeutics for HCC treatment.
4. Materials and methods
Gene chip analysis and human HCC samples
99 HCC tumor tissues and matched non-tumor tissues from archived patients at
the Hepatic Surgery Centre, Tongji Hospital of Huazhong University of Science and
Technology (Wuhan, China) were collected between 2017 and 2018. The diagnosis of
HCC was confirmed by pathological examination and the differentiation status was
graded according to the Edmondson grading system. After surgical resection, samples
were stored at -80˚C immediately. Written informed consent was obtained from all
subjects, and this study was conducted in accordance with the Declaration of Helsinki
and approved by the Ethics Committee of Tongji Hospital, Huazhong University of
Science and Technology.
HCC tissues and adjacent non-tumor liver tissues were placed in a cryogenic
tank with dry ice and were delivered to the Shanghai Biotechnology Corporation. The
Agilent version 16.0 was applied to screen the gene expression profiles of DNA repair
pathways between HCC tissues and their adjacent liver tissues.
Immunohistochemical (IHC) staining assays
Formalin-fixed paraffin-embedded sections were deparaffinized in xylene and
rehydrated with an ethanol gradient. The sections were placed in citric acid antigen
repair buffer (Wuhan Goodbio Technology Corporation, Wuhan, China) and antigen
repair was performed by microwaving on a low setting for 10 min. Endogenous
peroxidases were blocked by 3% H2O2. Sections were then incubated overnight with
an anti-EXO1 antibody at 1:200 (abs115859, Absin, Beijing, China) at 4 .℃
Secondary antibodies were incubated and peroxidase activity was detected using the
EnVision kit (Dako, Glostrup, Denmark). Hematoxylin (Sigma Aldrich, St. Louis,
MO, USA) was used for nuclear counterstaining. IHC scores were calculated as the
positive staining intensity scores multiplied by the stained positive cells scores.
Overall scores of >6 and ≤6 were defined as high expression or low expression of
EXO1, respectively.
Cell culture
The HCC cell lines Hep3B, Huh7, SK-Hep1, Bel-7402, and SMMC7721 were
purchased from the China Center for Type Culture Collection (Wuhan, China), and
HCC cell lines MHCC-97H and HCC-LM3 were obtained from the Liver Cancer
Institute, Zhongshan Hospital, Fudan University (Shanghai, China). The HCC cell
line HLE was obtained from the Department of Molecular Biology, Peking University
(Beijing, China). The HCC cell line PLC, human fetal liver cell line HL-7702,
hepatoma cell line HepG2 and lentivirus packaging cell line 293T were purchased
from the cell bank of the Chinese Academy of Sciences (Shanghai, China). Cell lines
were cultured in Dulbecco’s Modified Eagle’s medium (DMEM, Hyclone, Utah,
USA) containing 10% fetal bovine serum (Gibco, Carlsbad, CA). All cell lines were
incubated in a humidified atmosphere with 5% carbon dioxide at 37 .℃
Generation of EXO1 overexpression and knockdown constructs and FOXP3
knockdown construct
Full-length EXO1 coding sequence (CDS) was purchased from Vigene
Biosciences (Shandong, China). EXO1 was subcloned into pLenti-CMV-GFP-Puro
(plasmid# 17448; Addgene, Cambridge, MA, USA), according to the manufacturer’s
instructions.
For EXO1 knockdown assays, three shRNA (shEXO1-TRCN0000039788,
shEXO1-TRCN0000039789, and shEXO1-TRCN0000010331) sequences were
obtained from the Sigma website (www.sigmaaldrich.com). All these shRNAs were
subcloned into the lentiviral vector pLKO.1 puro (plasmid # 8453; Addgene,
Cambridge, MA, USA) according to the manufacturer’s instructions. Either the
shEXO1 constructs, EXO1 overexpression constructs or vector control plasmids were
transfected into the 293T cell line by Lipofectamine 2000 (Invitrogen, Thermo Fisher
Scientific, MA, USA) using the lentivirus packaging system. The plasmids pMD2.G
and psPA×2 were gifts from Didier Trono (plasmids# 12259 and# 12260; Addgene).
The supernatant-containing lentivirus produced by transfected 293T cells was
collected 72 hours later. Collected lentiviral supernatants were filtered through a
0.45μm filter (Millipore, USA). Target cells were infected with filtered lentivirus and
8 μg/mL polybrene (Sigma-Aldrich) to generate stable cell lines; treatment with 8
μg/mL puromycin (Ann Arbor, MI, USA) for 7 days was used to screen the positive
cells.
For siFOXP3 transfection assays, three siRNA (siFOXP3-1 stB0001154A,
siFOXP3-2 stB0001154B, and siFOXP3-3 stB0001154C) sequences were purchased
from RiboBio (Guangzhou, China). About 3×105 HLF or Huh7 cells/well were seeded
into a 6-well plate and infected with 5 μL of siRNAs after adherence. Lipofectamine
3000 (Invitrogen) was used for transfection.
Western blot analysis
Total protein was extracted from the cells using RIPA lysis buffer. Protein
concentrations were measured with a BCA Protein Assay Kit (Thermo Fisher
Scientific, MA, USA). Protein lysates were separated by SDS-PAGE and transferred
onto a polyvinylidene difluoride (PVDF) membrane (EMD Millipore, Billerica, MA,
USA). The membrane was blocked in non-fat milk in Tris-buffered saline containing
0.1% Tween-20 for 2 h and incubated with primary antibody (EXO1, ab95012,
Abcam, Cambridge, MA, USA; β-actin, Cell Signaling Technology, Danvers, MA,
USA) at 4°C overnight. Then, membranes were incubated with secondary antibodies
and signals were detected by Bio-rad chemiluminescence system.
Cell proliferation assay
Cells were inoculated into 96-well plates with 1×103 cells/well, and 100 µL of
10% cell counting kit-8 (CCK-8, Dojindo, Kumamoto, Japan) solution was added into
each well at 0, 1, 2, 3, 4, and 5 days after cell adherence. The optical density (OD)
values were measured by an EXL-800 absorbance reader at a wavelength of 450 nm
at the indicated time points (after Cell Counting Kit-8 solution was added to the wells
for 2 hours).
Cell migration and matrigel invasion assays
Cell migration and invasion assays were performed using transwell chambers
(Corning, USA) with or without Matrigel (BD Biosciences, CA, USA). About
1.5×104-10×104cells in medium without FBS were seeded on transwell chambers with
or without Matrigel. Below the transwell chambers, DMEM containing 10% fetal
bovine serum (FBS) was added. An automated cell counter (Nexcelom Bioscience,
USA) was used to quantify the number of migratory or invasive cells. Six fields at a
magnification of ×200 were randomly selected for counting stained cells.
Cell colony formation
EXO1-knockdown or -overexpressing HCC cells (HLF, Huh7, Bel7402) were
plated in 6-well plates (1000 cells/well) and culture media was replaced every 3 days.
After 2-3 weeks when clones were visible to the naked eye, cells were fixed with 4%
polyformaldehyde for 15 minutes and stained with crystal violet (Sigma-Aldrich
Corporation) for 15 minutes. Photographs of the 6-well plates were taken, and the
differences were counted.
Xenograft tumorigenicity assays
Animal studies were approved by the Ethics Committee of Tongji Hospital,
Huazhong University of Science and Technology. All animal experiments were
performed under the guidelines of the Interdisciplinary Principles and Guidelines for
the Use of Animals in Research, Testing, and Education by the New York Academy
of Sciences, Ad Hoc Animal Research Committee. 5-week-old male BALB/c (nu/nu)
mice were obtained from the HFK BioScience Corporation (Beijing, China). All the
mice were bred under specific pathogen-free (SPF) conditions at the Laboratory
Animal Center of the Tongji Hospital. For subcutaneous tumorigenesis assays, HLF
vector- and HLF shEXO1-788-transfected cells (2×106) were suspended in 100 μL of
Dulbecco’s modified Eagle’s medium (DMEM) and injected subcutaneously into the
left and right back of nude mice, respectively. Nude mice were monitored after the
injection and sacrificed at day 30 after cell inoculation. The final tumor volumes were
calculated according to the equation: V (volume, mm3) = 0.5 × L (length, mm) ×W2
(width, mm2).
RNA extraction and quantitative real-time PCR
Total RNA was extracted using TRIzol Reagent (Takara, Dalian, China) and
reverse transcription was performed using a reverse-transcription PCR kit
(TIANGEN, Beijing, China) according to the manufacturer’s instructions.
Quantitative real-time PCR was carried out using SYBR Green PCR master mix
(TIANGEN, Beijing, China) on a Bio-Rad CFX system (Bio-Rad, Hercules, CA,
USA) with the following primers:
EXO1-forward: 5' - TGAGGAAGTATAAAGGGCAGGT -3';
EXO1-reverse: 5'- AGTTTTTCAGCACAAGCAATAGC - 3'.
FOXP3-forward: 5'- GTGGCCCGGATGTGAGAAG - 3';
FOXP3- reverse: 5'- GGAGCCCTTGTCGGATGATG - 3'.
GAPDH- forward: 5'- GGAGCGAGATCCCTCCAAAAT- 3'.
GAPDH- reverse: 5'- GGCTGTTGTCATACTTCTCATGG- 3'.
The Ct values of EXO1 were equilibrated to those of the internal control GAPDH.
Luciferase reporter assay
The region 2000 bp upstream of the EXO1 translational start site was amplified
from the genomic DNA of the Huh7 cell line by PCR. EXO1 promoter regions of
different lengths were subcloned into pGL4.17 (Promega, Madison, WI, USA). The
open reading frame of FOXP3 and other candidate genes was amplified by PCR and
then cloned into pcDNA 3.1(+) (Invitrogen, Thermo Fisher Scientific, MA, USA) to
produce a series of products such as pcDNA 3.1-FOXP3 and so on. pGL4.17 and
pcDNA3.1 were used as controls. pRL-TK was purchased from Promega (Madison,
WI, USA). About 1×105 Huh7 cells/well were seeded into a 24-well plate and co-
transfected with 4 ng of the pRL-TK, 200 ng of pgl4.17 and 400 ng of pcDNA3.1
after adherence. Lipofectamine 2000 (Invitrogen) was used for transfection. Cell
lysates were prepared using passive lysis buffer (Promega) 48 h after transfection. A
luciferase activity was performed using the Dual-Luciferase Reporter Assay System
(Promega, Madison, WI, USA) according to the manufacturer’s instructions. The
relative luciferase activity was measured using a GloMax 20/20 Luminometer
(Promega).
Data acquisition and processing
Data for bioinformatics analysis were collected from The Cancer Genome Atlas
(TCGA), Oncomine and Gene Expression Omnibus (GEO) databases. The cor.test ()
function in the R (version 3.5.2) was used to calculate and verify the correlation
coefficients between the EXO1 and FOXP3. Then R packages ggplot2 (version 3.1.1)
was used for plotting.
Statistical analysis
Statistical analyses were performed with IBM SPSS Statistics 21.0 (SPSS,
Chicago, IL, USA) or Prism 6.0 (GraphPad Software, La Jolla, CA) software. The
results were presented as the mean ± standard deviation (SD) or mean ± standard error
of mean (SEM). Quantitative data were compared by a two-tailed Student’s t-test,
One-way ANOVA and Mann-Whitney U test when applicable. Categorical data were
analyzed by the χ2 test or Fisher’s exact test. Kaplan-Meier survival analysis (log-
rank test) was used to compare HCC patient survival. A two-sided value of p<0.05
was considered statistically significant.
Results
EXO1 is an upregulated DNA damage repair gene in HCC
In order to screen the expression of DNA damage repair genes, three samples of
HCC tissues and adjacent non-tumor tissues were collected for whole-genome
sequencing. Gene chip analysis showed that 33 DNA damage repair genes were
expressed at higher levels in tumor tissues compared to their corresponding non-
tumor tissues (Figure 1), and in tumor tissues, 8 of them were expressed at levels
greater than 10 times the normal levels. The expression of EXO1 in HCC tumor
tissues was 27.2 times higher than that in peri-cancerous liver tissues (Table 1).
Hence, we chose to perform follow-up studies on EXO1 and focused on its functional
roles and mechanisms EXO1 in driving HCC development and metastasis.
Clinical significance of EXO1 in HCC patients
First, we investigated EXO1 expression in tumor and non-tumor liver tissues in
three different clinical studies from the Oncomine database (Figure 2A). The results
showed that the expression of EXO1 was significantly higher in tumor tissues than
that in their adjacent non-tumor tissues. Next, the expression level of EXO1 was
detected in 99 HCC samples from Tongji Hospital (Wuhan, China) by western
blotting (Figure 2C, D). These results showed that the expression of EXO1 in HCC
tumor tissues was higher (79%) than that in adjacent non-tumor tissues (21%) (Figure
2B). We further explored the relationship between clinicopathological features and the
expression level of EXO1 in HCC patients. Ninety-nine HCC patients were divided
into an EXO1 low-expression group or EXO1 high-expression group. The detailed
information is summarized in Table 2. High expression of EXO1 was significantly
associated with the presence of liver cirrhosis (p=0.005). However, the expression of
EXO1 was not correlated with gender, age, or AFP level. We then used the TCGA
database to analyze the relationship between EXO1 expression and long-term
prognosis of HCC. Kaplan-Meier analysis revealed that the disease-free survival rate
and overall survival rate of patients with high EXO1 expression were significantly
lower than that of patients with low EXO1 expression (Figure 2E). Subsequently, the
expression level of EXO1 was assessed by immunohistochemistry (IHC) in 10 pairs of
tumor tissues and their adjacent non-tumor tissues. As shown in Figure 2F, EXO1 in
hepatocytes was localized to the cytoplasm, and the average IHC score of EXO1 in
tumor tissues was significantly higher than that in adjacent non-tumor tissues. Taken
together, these results indicate that EXO1 expression is significantly upregulated in
HCC tissues and high expression of EXO1 plays an important role in the prognosis of
HCC patients and may contribute to the progression of HCC.
EXO1 enhances proliferation and colony formation of HCC cells in vitro, as
well as tumor growth in vivo
To study the tumorigenic ability of EXO1 in vitro and in vivo, western blot was
used to detect the expression level of EXO1 in 10 different HCC cell lines and one
immortal liver cell line, HL-7702 (Figure 3A). The results showed that EXO1 was
upregulated in HLF and Huh7 cell lines, but downregulated in the Bel-7402 cell line.
Hence, three short hairpin RNAs (shEXO1-788, shEXO1-789, and shEXO1-331)
specifically targeting EXO1 were stably transfected into HLF and Huh7 cell lines, and
an empty vector was transfected into each cell line (abbreviate as vector) and used as
negative controls. The EXO1-shRNA significantly reduced EXO1 protein expression
in HLF and Huh7 cells compared with their control vectors in the western blotting
results. This result was also verified by RT-PCR (Figure 3B). EXO1 was also stably
transduced into Bel-7402 cell lines and an empty vector was used as a control.
Western blot and RT-PCR verified that EXO1 was significantly overexpressed in
transfected Bel-7402 cells (Figure 3C). Using functional assays, the tumorigenicity of
EXO1 was investigated. The Cell counting kit-8 (CCK-8) assay showed that EXO1
knockdown in HLF and Huh7 cells significantly inhibited cell proliferation, while
EXO1 overexpression in Bel-7402 cells markedly promoted cell proliferation
compared with their control vectors (p<0.01, Figure 3D, E). In the colony formation
assay, the colony-forming ability of EXO1-knockdown cells was markedly lower than
that of their control vectors (p<0.01, Figure 3F), whereas EXO1-overexpression in the
cells showed significantly higher colony-forming ability than in the control vectors
(p<0.01, Figure 3G). For in vivo tumorigenicity assays, HLF vector (control) and
HLF-shEXO1-788-transfected HCC cells were injected subcutaneously into nude
mice (Figure 3H). As expected, the final tumor weights and volumes of the HLF-
shEXO1-788 cell-injected group were significantly reduced compared to that of the
HLF vector cell-injected group (p=0.03, Figure 3I, J). These results indicate that
EXO1 has a strong promotive effect on cell proliferation and colony formation in
HCC cells, and even promotes HCC tumorigenicity.
EXO1 promotes tumor invasion and metastasis in vitro
Metastasis is one of the reasons for poor outcomes in HCC. To further explore
whether EXO1 can affect the invasion and metastatic abilities of HCC cells, we
performed transwell assays. As shown in Figure 4A, migratory abilities were
markedly decreased when EXO1 expression was downregulated by shRNA in HLF
and Huh7 cells compared to their respective controls. Additionally, as shown in Figure
4B, invasive abilities of HLF-shEXO1 and Huh7-shEXO1 were observably reduced.
In contrast, the migratory and invasive abilities of Bel7402-EXO1 cells
overexpressing EXO1 were much higher than their corresponding controls (Figure 4C,
D). Altogether, these results demonstrate that EXO1 plays an important role in the
invasion and metastasis of HCC.
Prediction of upstream regulators of EXO1 via bioinformatics analysis
To elucidate the molecular mechanisms of EXO1 overexpression in HCC tumor
tissues, we concentrated on the region 2 kb upstream of the transcription start site
(TSS) of EXO1 (Figure 5A). Introduction of this 2-kb region upstream of firefly
luciferase in pGL4.17-basic yielded significantly greater reporter activity compared
with the promoter-less construct in Huh7 cell lines (28-fold) (Figure 5B). With serial
truncations of the 2 kb sequence at 500-bp intervals, we generated a set of luciferase
reporter constructs (500 bp, 1000 bp, 1500 bp, 2000 bp). A luciferase reporter assay
showed a remarkable rise in luciferase activity when the upstream sequence of TSS of
EXO1 was 1000 bp long, and its luciferase activity was the strongest of the four
sequences (p<0.05, Figure 5B). To further determine whether there is a region with
higher transcriptional activity involved in the 1000-bp sequence, we continued to
generate luciferase reporter constructs with truncations of the 1000-bp sequence at
200-bp intervals, which were 200 bp, 400 bp, 600 bp, and 800 bp upstream of the TSS
of EXO1. As shown in Figure 5C, the relative reporter activity of the 1000-bp
sequence was still the highest compared to other sequences. These results suggest that
the 1000-bp region directly upstream of the TSS of EXO1 possesses the highest
transcriptional activity, and transcription factors possibly bind to the 1000-bp segment
of EXO1 promoter.
FOXP3 could influence the expression of EXO1 in HCC
To identify the transcription factors that could bind to the promoter region of
EXO1, we used the PROMO and JASPA websites to make predictions. According to
the scoring system provided by the website and gene function itself, we focused on
four transcription factors named FOXP3, E2F2, GATA2 and HNF1A. Plasmids were
constructed with four coding sequences of these transcription factors mentioned above
using pcDNA3.1 as a vector. We detected the transcriptional activity of these four
transcription factors in the Huh7 cell line by luciferase reporter assay. Interestingly, as
Figure 6A shows, FOXP3 had a significantly high reporter activity level compared to
the other transcription factors. Based on this crucial result, we performed the same
luciferase reporter assay in the Hep3B and PLC cell lines. As shown in Figure 6B and
6C, compared with co-transfection of the pcDNA 3.1 vector and pGL4.17-1000, when
co-transfected with pcDNA 3.1-FOXP3 and PGL 4.17-1000, there was strong reporter
activity. These results strongly suggest that FOXP3 may be involved in the
transcriptional regulation of the EXO1 gene. Through bioinformatics analysis in the
GEO database GSE109211, we found that the expression of FOXP3 and EXO1 had a
positive correlation in HCC (Figure 6D). Hence, we transiently transfected the
pcDNA3.1-FOXP3 plasmid into the three different HCC cell lines (Huh7, PLC,
Hep3B) and transfected a pcDNA3.1 vector as a control. Consistently, the mRNA
level of EXO1 increased with FOXP3 transfection (Figure 6E), as detected by RT-
PCR. Together, these results show that FOXP3 promotes the expression of EXO1
mRNA levels and may regulate the expression of EXO1.
Depletion of FOXP3 enhances the proliferation effects of EXO1 in vitro
Due to the regulatory impact of FOXP3 on EXO1, we wanted to explore whether
FOXP3 can affect the proliferation activity of EXO1 in HCC cells. RT-PCR showed
that among the three siFOXP3 knockdown sequences, FOXP3 was significantly
downregulated by siFOXP3-1 in HLF and Huh7 cell lines (p<0.05, Figure 7A, B).
Next, we transfected siFOXP3-NC into shEXO1-vector HCC cells and shEXO1-788
HCC cells, respectively, and transfected siFOXP3-1 into shEXO1-788 HCC cells. As
shown in Figure 7C and D, CCK-8 assays indicated that compared with HCC cells
with only EXO1 knockdown, FOXP3 and EXO1 double knockdown significantly
decreased the proliferation ability of the HCC cells (p<0.05). These results suggest
that depletion of FOXP3 further reduces cell proliferation ability after knocking down
of EXO1 in vitro, suggesting that FOXP3 may upregulate EXO1, and depletion of
FOXP3 may reduce its expression.
Discussion
It has been shown that DNA damage caused by the hepatitis B virus (HBV)
infection is one of the main molecular mechanisms of hepatocellular carcinoma [35-
37]. HBV-DNA can be integrated into host genomic DNA and cause insertion
mutations or other genomic instabilities [38, 39]. Studies have shown that DNA
double strand breaks (DSBs) induced by DNA damage are potential targets for HBV-
DNA integration [40-42]. The body repairs DSBs through two major repair pathways:
homologous recombination (HR) and non-homologous end-joining (NHEJ) [43]. Bill
et al. previously suggested that integration of duck hepatitis B virus (DHBV) DNA
into the host hepatocyte genome may be accomplished by the body's NHEJ repair of
DSBs, and the integration could occur at the sites of DNA damage [44]. EXO1 plays
an important role in DNA damage repair, especially in HR and NHEJ after DSBs [45-
48]. As HR and NHEJ may participate in the integration of HBV-DNA, the
relationship between EXO1 and HBV related HCC is worth investigating. In the
present study we observed that EXO1 was frequently overexpressed in HCC samples
and the same result was also seen in the Oncomine database. Analysis of EXO1
prognosis from the TCGA database showed the overall survival rate and tumor-free
survival rate of patients with high EXO1 expression were significantly lower than
those of patients with low EXO1 expression. Thus, we concluded that EXO1 is highly
expressed in HCC and is associated with poor prognosis.
The link between EXO1 and cancer is still under continuous research and much
progress has been made. As mentioned earlier, several SNPs in the EXO1 gene are
related to the occurrence of certain tumors [17-25]. Some EXO1-specific mutations
such as E109K and A153V are thought to be located in the region required for
nuclease activity, thereby deactivating protein function and affecting cancer
susceptibility [49, 50]. In the mouse model, the incidence of lymphoma in EXO1null/null
increased and the survival rate decreased, but the microsatellite instability (MSI) was
not affected [13]. EXO1 has also been reported to be overexpressed in several other
cancers as described in the introduction [15, 16, 51-53]. In general, EXO1 is poorly
expressed and increased levels of EXO1 may cause cellular damage due to its 5'-3'
exonuclease activity, which may be contrary to previous studies [8, 54]. This may be
due to the complex role of EXO1 in DNA damage repair and its tissue-specific
expression. In humans, EXO1 is highly expressed in the testis and moderately
expressed in the thymus, colon, and placenta [5, 55]. The liver has high mitochondrial
activity and increased reactive oxygen species (ROS), which may lead to increased
activation of the DNA damage repair pathway, resulting in a relatively high
expression level of EXO1 in the liver [55]. Muthuswami et al. treated breast cancer
cell line, MCF7, with different alkylating agents such as carboplatin,
cyclophosphamide, etc. to induce DNA repair and found that EXO1 expression
increased with increasing concentration of these alkylating agents [51]. de Sousa et al.
observed that EXO1 knockdown supported a faster DNA-DSB restoration after
irradiation (IR) exposure in glioblastoma cell line T98G [15]. All these, explain to
some extent that EXO1 is involved in the DNA repair pathway under cancerous
conditions. The overexpression of EXO1 in tumors may also increase the genetic
instability and contribute to tumor initiation and progression through its involvement
in recombinant events such as DSBs and telomere stabilization [56].
At present, the research on the role of EXO1 in HCC is still lacking. Previously, it
was reported that SNPs K589E (rs1047840) and rs3754093 of EXO1 could increase
and decrease the susceptibility of HCC, respectively [17, 18]. In 2018, Dai et al. used
clonogenic assays to find that the expression of EXO1 affects the HCC cell survival
under irradiation, and EXO1 overexpression has a poor prognosis for HCC patients
[16]. In other types of tumors, as previously mentioned, Muthuswami et al. and de
Sousa et al. investigated the possible role of EXO1 in different types of tumors
through the breast cancer cell line MCF7 and the glioblastoma cell line T98G,
respectively [15, 51]. In 2019, Luo et al. found the differential expression of EXO1 in
five prostate cancer cell lines, and the EXO1 expression in the other four high‐
invasive/metastatic prostate cancer cell lines significantly increased compared with
the low‐invasive/metastatic potential cell line LNCaP [53]. These researches above
led us to explore the role of EXO1 in the malignant biological characteristics of HCC
by using HCC cell lines in vitro and in vivo. In this study, we observed that
knockdown of EXO1 reduced cell proliferation in vitro and in vivo, while
overexpression of EXO1 enhanced it. Subsequently, transwell assays showed that
EXO1 could markedly increase the cell motility of HCC cells in vitro, indicating that
EXO1 can affect the migration and invasion of HCC cells. Altogether, these functional
assays indicate that EXO1 may play a carcinogenic role in HCC. However, previous
research reported that knockdown of EXO1 in mice exhibits a mild phenotype, which
may be contrary to our experimental results [57]. This may be due to complex
environmental factors in vivo as well as the multiple roles the EXO1 gene itself play
in DSBs. There are still some researches suggesting that the absence of EXO1 in mice
will show MMR defects, apoptotic defects, and sterility [10, 11]. Therefore, although
we suggest that EXO1 may function as a tumorigenic gene in vitro, more in vivo
experiments are needed to validate these conclusions.
EXO1 has been previously reported to be regulated by a variety of molecules and
to bind to certain specific proteins [58, 59]. Studies on DNA mismatch repair (MMR)
have reported that EXO1 interacts with the mismatch repair factors mutL homolog 1
(MLH1), mutS homolog 2 (MSH2), mutS homolog 3 (MSH3), and proliferating cell
nuclear antigen (PCNA) to excise the newly synthesized DNA sequences containing
errors during the MMR process [60-63]. Checkpoint regulatory proteins 14-3-3 are
widely reported as interaction partners of EXO1. Engels et al. identified in 2011 that
in yeast and mammalian cells, 14-3-3 proteins interact with EXO1 in vivo to regulate
the phosphorylation status of EXO1, thereby promoting EXO1-dependent fork
progression after inhibiting replication [64]. In 2012, Andersen et al. demonstrated
specific interactions between EXO1 and all the 7 isoforms of 14-3-3 through in vitro
GST pull-down assays, and they demonstrated that the binding may involve a second
unrecognized binding motif in EXO1, the amino acid S746. They also demonstrated
that the 14-3-3 association may affect the nuclease activity of EXO1. The nuclease
activity of EXO1 can also be affected by the 14-3-3 association [65]. In 2015, Chen et
al. showed that 14-3-3 can interact with the central portion of EXO1, at least partially,
by suppressing its association with PCNA, to negatively regulate EXO1 damage
recruitment and thus prevent excessive DNA excision [66]. However, very little is
known about the transcriptional regulation of EXO1. Muthuswami et al. discovered
that transcription factors E2F and Myc may be involved in the regulation of EXO1 by
using luciferase reporter assays [51]. In this study, we uncovered a new potential
transcriptional regulator of EXO1, FOXP3, by truncating the promoter region of
EXO1 and using bioinformatic prediction. FOXP3 has been proposed to function as a
tumor suppressor in breast and prostate epithelial cells [67, 68]. However, FOXP3
could also be a prognostic factor in breast cancer and play an important role in the
progression of cervical cancer [69, 70]. In a recent report from 2019, Ou et al.
reported that FOXP3 silencing may be associated with the inhibition of cell
proliferation and migration of HCC cells, as well as the induction of apoptosis [71].
By transiently transfecting FOXP3 into HCC cell lines, we observed that FOXP3
could promote the transcriptional activity of EXO1 by a luciferase reporter assay. We
further found that EXO1 mRNA levels were significantly upregulated after transient
transfection of FOXP3. Moreover, bioinformatics analysis revealed a positive
correlation between the expression of EXO1 and FOXP3, and a CCK-8 assay in HCC
cells proved that FOXP3 could enhance the proliferative effect of EXO1 in vitro.
These results indicate that FOXP3 could upregulate the transcription of EXO1 and
might further regulate the expression level of EXO1. This is consistent with the
reported results of Ou et al. Further explorations are required to validate this
perspective.
Nonetheless, this study has some limitations. Firstly, the size of the clinical
samples in this study was relatively small, with only 99 cases. Therefore, the number
of samples assessed needs to be expanded. Second, there were database limitations
and the expression level of EXO1 and the prognosis of HCC patients need to be
further verified by complete clinical follow-up. Finally, it is not uncommon that DNA
repair genes are highly overexpressed in HCC. In addition to the possible
explanations given above, X-Ray Repair Cross Complementing 2 (XRCC2) has also
been reported to be up-regulated in a variety of tumors [72, 73]. It is possible that
DNA damage and repair processes are more active in tumor cells. RAD51B,
RAD51C, RAD51D, and XRCC2 form the BCDX2 complex, which promotes the
aggregation of RAD51 protein at HR sites [74]. The mechanism of EXO1
overexpression in HCC is complex and still needs to be further explored.
In summary, our research found that EXO1 was overexpressed in HCC and
played a carcinogenic role in HCC, and that FOXP3 may upregulate the expression
level of EXO1. For the first time, we have uncovered a possible transcriptional
regulation mechanism of EXO1. This discovery provides clues for the relationship
between DNA damage repair genes and HCC and might lead to the development of
novel anti-cancer therapeutics for HCC treatment.
Abbreviations
EXO1: Exonuclease 1; FOXP3: Forkhead box P3; HCC: Hepatocellular
carcinoma; TNM: Tumor-node-metastasis; AFP: α-fetoprotein; ALT: alanine
aminotransferase; AST: aspartate aminotransferase; SD: Standard deviation; SEM:
Standard error of mean
Author contributions
Guang Yang performed the experiments and wrote the article independently. Ke-
Shuai Dong conducted the bioinformatics analysis and did the statistical analysis.
Zun-Yi Zhang provided technical guidance for the entire study. Er-Lei Zhang, Bin-
Yong Liang collected and analyzed the clinical data. Zhi-Yong Huang directed the
revision and correction of the manuscript. Xiao-Ping Chen and Zhi-Yong Huang
conceived the idea and designed the study. All authors reviewed and endorsed the
final manuscript.
Abbreviations:
EXO1, Exonuclease 1; FOXP3, Forkhead box P3; HCC, Hepatocellular
carcinoma; TNM stage, Tumor-node-metastasis stage; AFP, α-fetoprotein; SD,
Standard deviation; SEM, Standard error of mean
Acknowledgments
This work was funded by the National Natural Science Foundation of China
(81172293) and the National Science and Technology Major Project of China
(2017ZX10203207-002).
Conflicts of interest
The authors have no conflict of interest to declare.
References
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Figure 1. EXO1 is an up-regulated DNA damage repair gene in HCC. Heat Map
of top-ranked upregulated DNA damage repair genes in HCC tissues compared with
adjacent normal liver tissues detected by gene chip technology. The results showed
that the expression level of 33 molecules in HCC tissues was higher than that in
corresponding non-tumor tissues. (P: Pericancerous liver tissue; C: hepatocellular
carcinoma tissue)
Table 1. Expression of DNA damage repair molecules increased by more than 10-fold
GenBank
accession
Gene
name
Description Fold
change
NM_130398 EXO1 Exonuclease 1 27.2*
NM_005431 XRCC2 X-ray repair complementing defective repair in Chinese
hamster cells 2
17.5
NM_001789 CDC25A Cell division cycle 25 homolog A 15.8
NM_002875 RAD51 RAD51 homolog 13.8
NM_001274 CHEK1 CHK1 checkpoint homolog 13.6
NM_032043 BRIP1 BRCA1 interacting protein C-terminal helicase 1 11.4
NM_012238 SIRT1 Sirtuin 1 11.4
NM_001790 CDC25C Cell division cycle 25 homolog C 10.8
Figure 2. EXO1 expression is upregulated in HCC and associated with poor
outcomes in HCC patients. (A) Dot chart of analysis of EXO1 expression in HCC
tumor tissues and non-tumor tissues in the Oncomine database. (B) The expression of
EXO1 in 99 paired HCC tissues and adjacent non-tumor tissues was detected by
western blotting. The bar chart shows the expression level of EXO1 in HCC tissues,
which were quantified and normalized to the corresponding EXO1 levels in the
adjacent non-tumor tissues. (C) Dot chart of EXO1 expression in the 99 HCC
samples. (D) Representative images of Western blots of EXO1 in 99 HCC tumor
tissues and their adjacent non-tumor tissues. (E) Kaplan-Meier analysis showed the
disease-free survival curve and overall survival curve of two different groups: patients
with high EXO1 expression and patients with low EXO1 expression. The data comes
from TCGA database. (***p<0.001). (F) IHC analysis of EXO1 expression in 10
paired HCC tissues. Results of case 1 and case 3 are used as representative images in
the left panels and statistical analysis of EXO1 expression in the HCC samples in the
right panel. (20×, magnification) (*p<0.05; **p<0.01; *** p<0.001).
Table 2. The correlation between EXO1 expression and clinicopathological features in HCC
Clinical variablesNo. of patients EXO1 expression level
p valuen=99 Low(n=21) High(n=78)
Age(years) 0.118 <60 66 17(81.0%) 49(62.8%) ≥60 33 4(19.0%) 29(37.2%)Gender 0.123 Male 87 21(100.0%) 66(84.6%) Female 12 0(0.0%) 12(15.4%)HBsAg 1.000 Positive 88 19(90.5%) 69(88.5%) Negative 11 2(9.5%) 9(11.5%)AFP(ng/ml) 0.911 <400 67 14(66.7%) 53(67.9%) ≥400 32 7(21.9%) 25(78.1%)Child-Pugh Class 0.152 A 88 21(100.0%) 67(85.9%) B 11 0(0.0%) 11(14.1%)Liver cirrhosis 0.005 No 19 9(42.9%) 10(12.8%) Yes 80 12(57.1%) 68(87.2%)Tumor size(cm) 0.709 ≤5 46 9(42.9%) 37(47.4%) >5 53 12(57.1%) 41(52.6%)Tumor number 0.589 Single 86 17(81.0%) 69(88.5%) Multiple 13 4(19.0%) 9(11.5%)Tumor encapsulation 0.227 Yes 63 11(52.4%) 52(66.7%) No 36 10(47.6%) 26(33.3%)Vascular invasion 0.094 Yes 22 8(38.1%) 14(17.9%) No 77 13(61.9%) 64(82.1%)Tumor differentiation 0.903 Well 12 2(9.5%) 10(12.8%)Middle 52 11(52.4%) 41(52.6%)
Poor 35 8(38.1%) 27(34.6%)TNM stage 0.531 I-II 75 17(81.0%) 58(74.4%) III-IV 24 4(19.0%) 20(25.6%)
Figure 3. EXO1 promotes cell proliferation and colony formation in HCC cell
lines and proliferation in vivo. (A) The expression levels of EXO1 in one healthy liver
cell line, ten HCC cell lines and one HCC lung metastasis cell line were detected by
western blotting. (B), (C) Western blotting confirmed that EXO1 expression was
effectively repressed by shRNA in HLF and Huh7 cell lines and EXO1 was
overexpressed in the Bel-7402 cell line. The bar chart shows that EXO1 mRNA levels
were significantly decreased or increased in the corresponding cell lines compared to
the vector group. The data are shown as the mean ± SEM. (D), (E) CCK-8 assays
indicated that knockdown of EXO1 significantly reduced cell proliferation abilities
while overexpression of EXO1 increased cell proliferation abilities in the
corresponding cell lines. The data are shown as the mean ± SD (n=6) (F), (G)
Representative image of colony formation assays of EXO1 knockdown and EXO1
overexpression in the corresponding cell lines. The bar chart shows that EXO1
knockdown reduced colony formation abilities but overexpression of EXO1 enhanced
colony formation abilities compared to their vector groups. Data are represented as
the mean ± SD (n=3). (H) Representative image of subcutaneous tumors (n=5). (I)
Dot chart of final tumor weight (n=5). (J) Dot chart of final tumor volume (n=5).
(*p<0.05; **p<0.01; ***p<0.001 when compared with the vector group).
Figure 4. EXO1 promotes migration and invasion of HCC cells in vitro. (A), (B)
Representative images of migration and invasion of HLF and Huh7 cells by transwell
migration and matrigel invasion assays. Bar chart shows that migration and invasion
abilities were significantly reduced when EXO1 was downregulated compared to the
vector groups. The data are shown as the mean ± SD (n=6). Scale bar: 200 μm. (B),
(C) Representative images of migration and invasion of Bel-7402 cells by transwell
migration and matrigel invasion assays. The bar chart shows the migration and
invasion abilities were significantly increased when EXO1 was overexpressed
compared to the vector groups. The data are shown as the mean ± SD (n=6). Scale
bar: 200 μm (***p<0.001 when compared to the vector group).
Figure 5. Truncation of EXO1 promoter region. (A) Schematic diagram of the
region lying 2000 bp upstream of EXO1 transcription start site and a series of
truncations. (B) Schematic diagram of the serial 500- promoter truncations (red)
placed upstream of the EXO1 translational start site and their corresponding luciferase
reporter activities. Data are represented as the mean ± SD (n=3). (C) Schematic
diagram of the serial 200- promoter truncations (red) placed upstream of the EXO1
translational start site and their corresponding luciferase reporter activity. Data are
represented as the mean ± SD (n=3). (*p<0.05).
Figure 6. FOXP3 enhances the expression of EXO1. (A) Bar chart of luciferase
activity of transcription factors possibly binding to EXO1 promoter region compared
to the pcDNA3.1-vector group. Data are represented as the mean ± SD (n=3). (B), (C)
pcDNA3.1 and pGL4.17 were co-transfected into Hep3B and PLC cells for 48 h. The
bar chart of luciferase activity shows that when co-transfected with pcDNA3.1-
FOXP3 and pGL4.17-1000, the relative reporter activity was strongest. Data are
represented as mean ± SD (n=3). (D) The scatterplot diagram of positive correlation
between EXO1 and FOXP3 expression level in GSE109211 (n=140). (E) pcDNA3.1-
FOXP3 or pcDNA3.1-vector were transiently transfected into Huh7, PLC, and Hep3B
cells for 48 h. The expression of EXO1 was examined by RT-PCR. Data are
represented as mean ± SD (n=3). (*p<0.05; **p<0.01; ***p<0.001).
Figure 7. Depletion of FOXP3 enhances the proliferative effects of EXO1 in vitro
(A), (B) RT-PCR confirmed that FOXP3 expression was effectively repressed by
siFOXP3-1 in HLF and Huh7 cell lines. The data are shown as the mean ± SEM. (C),
(D) CCK-8 assays indicated that knockdown of FOXP3 in EXO1 knockdown cells
significantly reduced cell proliferation abilities compared to the EXO1 knockdown
alone. The data are shown as the mean ± SD (n=6). (*p<0.05; **p<0.01;
***p<0.001).